Immunoassays, which take advantage of natural immunological reactions, have found widespread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes which are present in very low concentration and cannot be adequately quantitated by chemical techniques. Such analytes (called ligands herein) include, for example, therapeutic drugs, narcotics, antibiotics, hormones, proteins, and others known in the art. Several techniques have been devised for determining very low concentrations of ligands. For instance, a ligand may be labeled by various means to make it readily detectable. In competitive binding assays, a labeled ligand analog (identified as ligand analog herein) is placed in competition with unlabeled ligand for reaction with a fixed amount of the appropriate binding material (also called a receptor). Unknown concentrations of the ligand can be determined from the measured signal of either the bound or unbound (that is, free) ligand analog.
Sensitivity is of prime importance due to the extremely low levels of ligands to be determined. While a variety of labels can be used, fluorescent or enzyme labels are generally preferred in most immunoassays due to increased sensitivity.
Immunoassays can also be classified as either heterogeneous or homogeneous. Heterogeneous competitive binding immunoassays require a separation of bound ligand analog from free ligand analog. This separation is necessary because the properties of bound and free analog are not significantly different. Homogeneous immunoassays do not require a separation step because the properties of the bound and free analogs are sufficiently different so that they can be readily differentiated.
U.S. Pat. No. 4,670,381 (issued Jun. 2, 1987 to Frickey et al) describes a multilayer analytical element which can be used for immunoassays. This element comprises a porous spreading layer in which a receptor (for example, an antibody) for the ligand to be determined is immobilized. This receptor is immobilized on a suitable carrier material, such as glass or polymeric beads or a microorganism such as Staphylococcus aureus. Alternatively, the beads used to form the porous spreading layer can be used to immobilize the receptor.
Biologically active polypeptides or proteins which are attached to insoluble carrier materials, such as polymeric particles, have been used in a variety of ways in immunoassays. For example, the diagnosis of pathological or other conditions in human beings and animals is often carried out using immunological principles for the detection of an immunologically reactive species, for example antibodies or an antigen, in the body fluids of the person or animal. An antigen is generally known as a foreign substance, such as a drug, hapten, toxin, lectin, glycoprotein, polysaccharide, glycolipid, polypeptide or protein which, when introduced into the body, causes the production of certain soluble proteins known as antibodies.
Other proteins, such as enzymes, have been covalently linked to various carrier materials for use in affinity chromatography, enzymatic reactions, specific binding reactions and immunoassays. Among useful carrier materials are sheep and human erythrocytes, bacterial cells, latex particles, resinous particles and finely divided diazotized amino cellulose. For example, carrier particles prepared from sparingly water-soluble monomers (such as epoxy group-containing monomers) in the absence of emulsifiers are described in U.S. Pat. No. 4,415,700 (issued Nov. 15, 1983 to Batz et al).
Carboxylated latex particles have also been used to prepare diagnostic reagents as described, for example, in U.S. Pat. No. 4,181,636 (issued Jan. 1, 1980 to Fischer). As described therein, the conventional procedure for covalently attaching an immunologically reactive species to the particles having surface carboxyl groups involves the use of a water-soluble carbodiimide in an additional activation step. While producing useful reagents, this procedure tends to activate the exposed reactive groups of the reactive species as well as the carboxyl groups. The result is intramolecular and intermolecular crosslinking or polymerization of the immunologically reactive species, and a significant portion of the species is thus impaired from complexation with a receptor molecule. Because the reactive species, for example an antibody, is usually very costly, this problem represents a serious economic loss. Further, sensitivity of the resulting reagent may be impaired. It has also been evident that carbodiimides provide a reactive intermediate for protein attachment which is unstable and must be used immediately. This can be a serious drawback to carbodiimide chemistry.
Various other reagents have been prepared with particles having reactive groups such as epoxides, aldehydes, chloromethyl groups, amine groups and diazonium salts. All of these groups have their disadvantages. For example, epoxide groups are not stable so that the particles cannot be stored for very long. Particles having aldehyde groups generally tend to agglutinate prematurely. particles with amine groups are like the carboxylated materials by requiring an additional activation step. Diazonium compounds are unstable and therefore undesirable to work with.
U.S. Pat. No. 4,283,382 (issued Aug. 11, 1981 to Frank et al) describes immunoreactive reagents having europium chelate tracer materials within the particles. Some of the reagents are prepared from polymers having reactive chloromethyl groups (see Col. 7, lines 28-32). While such materials provide some advantages, attachment of the immunological species to such polymers requires elevated temperatures, extended reaction time and acute mixing conditions. If the attachment conditions are not just right, attachment is incomplete, resulting in a reagent with poor sensitivity.
Reagents which are composed of a protein attached to a water-insoluble particle, then are very useful in a number of methods, including immunoassays, diagnostic methods and the like. It would be very useful if highly sensitive reagents could be readily prepared in an efficient manner and under conditions which are not limiting and which do not reduce sensitivity or generate other undesirable results.